Cytochrome P450 (P450) is a term used for a widely distributed group of unique heme proteins which form carbon monoxide complexes with a major absorption band at wavelengths around 450 nm. These proteins are enzymes which carry out oxidase functions in a wide variety of mixed function oxidase systems involved in biosynthesis and catabolism of specific cell or body components, and in the metabolism of foreign substances entering organisms. Oxygenating enzymes such as P450 appear to be fundamental cellular constituents in most forms of aerobic organisms. The activation of molecular oxygen and incorporation of one of its atoms into organic compounds by these enzymes are reactions of vital importance not only for biosynthesis, but also for metabolic activation or inactivation of foreign agents such as drugs, food preservatives and additives, insecticides, carcinogens and environmental pollutants.
In eukaryotic systems P450, and P450 dependant enzymes are known to act on such xenobiotics and pharmaceuticals as phenobarbitol, antipyrine, haloperidol and prednisone. Known substrates of environmental importance include compounds such as DDT, and a variety of polychlorinated biphenyls and polyaromatic hydrocarbons, as well as other halogenated compounds, including halobenzenes and chloroform.
Hexamethylphosphoramide (HMPA) is a compound that was used heavily by industry in the mid-1970's in the production of aramid fibers and as a general solvent. HMPA is a known carcinogen and has been found to be one of the contaminants at various industrial and chemical waste sites. Studies focusing on the mammalian biodegradation of HMPA are few but it has been found that microsomal P450 isolated from rat liver and nasal mucosa will demethylate HMPA. Longo et al., Toxicol. Lett. 44:289 (1988).
In microbial systems cytochrome P450 is known to oxidize many of the same xenobiotic substrates as in eukaryotic systems and thus can be targeted as possible indicators for the presence of toxic compounds in the environment. One of the earliest reports of xenobiotic transformation was by the bacterium Streptomyces giseus which is known to contain the gene for the expression of cytochrome P450. This transformation involved the convention of mannosidostreptomycin to streptomycin. Sariaslani et al., Developments in Industrial Microbiology 30:161 (1989). Since then these reactions have been observed with compounds ranging from simple molecules such as benzene to complex alkaloids (such as vindoline and dihydrovindolin, codein, steroids, and xenobiotics such as phenylhydrazine, ajmaline and colchine. Sariaslani et al., Developments in Industrial Microbiology 30:161 (1989).
Genetically engineered microorganisms with the ability to express the P450 gene offer several potential advantages. Such microorganisms might be designed to express precisely engineered enzymatic pathways that can more efficiently or rapidly degrade specific chemicals. Development efforts are aimed largely at chemicals that are toxic or recalcitrant to naturally occurring bacterial degradation.
It has been shown that bacteria of the genus Streptomyces, when properly induced, are capable of producing both cytochrome P450soy and the iron-sulfur protein (ferredoxin-soy) that donates electrons to cytochrome P450soy. Sariaslani et al., Biochem. Biophys. Res. comm. 141:405 (1986) The induction procedure involves growing the bacteria in a medium comprising an inducer such as soybean flour, genistein or genistin.
The method of Sariaslani et al. for producing P450 is useful however, the need to utilize an inducer such as soybean flour or a soybean flour-like substance to induce production of cytochrome P450soy in bacteria of the genus Streptomyces is a drawback. Such inducers are difficult to work with and represent an unknown variable in the field. Also, the need to induce the bacteria to produce the desired enzyme introduces an additional step in the method, making the method more complex.
There is a need for a simple method of bioremediating methylated phosphoric amides such as HMPA without the use of inducers to stimulate enzymatic activity. A simple method would be based on the use of bacteria capable of constitutive expression of cytochrome P450soy and the iron-sulfur protein that donates electrons to cytochrome P450. The cytochrome P450soy enzyme in Streptomyces griseus bears a resemblance in its oxidative reactions to the cytochrome P450 enzymes of mammalian liver microsomes and thus Streptomyces griseus could serve as an economical and convenient source of cytochrome P450 for indication of the presence of hazardous chemicals as well as their possible bioremediation.